Process for preparing antibiotic EM 4940

ABSTRACT

Cultivation of a strain of the microorganism Nocardia sp. 11,340, which has been deposited in the American Type Culture Collection as A.T.C.C. No. 31531, yields a novel antibiotic substance EM 4940, having activity against a range of gram-positive and gram-negative bacteria, yeasts, fungi, acholeplasma, and the protozoan Trichomonas vaginalis.

This is a division of application Ser. No. 70,287, filed Aug. 27, 1979, now U.S. Pat. No. 4,249,008, issued Feb. 3, 1981.

SUMMARY OF THE INVENTION

Cultivation of a strain of the microorganism Nocardia sp. 11,340, which has been deposited in the American Type Culture Collection as A.T.C.C. No. 31531, yields a novel antibiotic substance EM 4940, having activity against a range of gram-positive and gram-negative bacteria, yeasts, fungi, acholeplasma, and the protozoan Trichomonas vaginalis.

The chemical structure of EM 4940, the antibiotic of this invention, has been elucidated through the use of x-ray crystallography and is as follows: ##STR1## i.e., 3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide. EM4940 is a mixture of two antibiotics, a 2,4-trans isomer and a 2,4-cis isomer, which have been designated EM 4940A and EM 4940B respectively.

DETAILED DESCRIPTION OF THE INVENTION The Microorganism

The microorganism used for the production of EM 4940 is a strain of Nocardia sp. SC 11, 340. A subculture of the organism can be obtained from the permanent collection of the American Type Culture Collection, Rockville, Maryland. Its accession number in this repository is A.T.C.C. No. 31531. In addition to the specific microorganism described and characterized herein, it should be understood that mutants of the microorganism (e.g., mutants produced through the use of x-rays, ultraviolet radiation or nitrogen mustards) can also be cultivated to produce EM 4940.

Nocardia sp. 11,340, A.T.C.C. No. 31531, can be isolated from a moist soil sample containing the microorganism by first stamping the soil sample on a medium containing:

    ______________________________________                                         KNO.sub.3            1      g.                                                 K.sub.2 HPO.sub.4    0.5    g.                                                 MgSO.sub.4 . 7H.sub.2 O                                                                             0.5    g.                                                 NaCl                 0.5    g.                                                 FeSO.sub.4 . 7H.sub.2 O                                                                             0.01   g.                                                 Glucose              5      g.                                                 Agar                 20     g.                                                 Distilled water      1      liter                                              ______________________________________                                    

The medium is sterilized in an autoclave at 121° C. for 20 minutes. After 14 days incubation at 28° C., colonies of the Nocardia sp. SC 11,340 are isolated from the plated soil. These isolated colonies are then grown on a medium containing:

    ______________________________________                                         Yeast extract          1     g.                                                Beef extract           1     g.                                                NZ Amine A             2     g.                                                (a casein hydrolysate)                                                         Glucose                10    g.                                                Agar                   15    g.                                                Distilled water        1     liter                                             ______________________________________                                    

The medium is adjusted to pH 7.3 and autoclaved at 121° C. for 30 minutes.

Nocardia sp. Sc 11,340 produces a vegetative mycelium which fragments into rod and coccal forms within 5 days. The cells are nonacid fast and gram-positive.

Colonies growing on a solid medium are smooth to doughly in consistency and are whitish gray in color. On glycerol asparagine agar there are no distinguishing characteristics; on oatmeal tomato paste agar, a soluble purple pigment is produced that permeates throughout the solid medium. No aerial mycelium is formed.

Acid hydrolysates of whole cell walls analyzed by the method of Becker et al., Applied Microbiology, 12:421 (1964), indicate the presence of mesodiaminopimelic acid, galactose and arabinose as the major sugar components found. There is some ribbose present but no mudurose. This cell wall analysis represents a Type, IV A pattern (Lechevalier et al, In: The Actionmycetales, H. Pransen (Ed.), VEB Guster Fisher Verlag, pgs. 398-400,402 (1970)) and is diagnostic for the genus Nocardia.

To determine whether acid products are formed from various carbohydrates in the presence of Nocardia sp. SC 11,340, the microorganism is cultured for 10 days on the basal medium of Ayers, Rupp and Johnson, Bull. U.S. Dept. Agri., No. 782 (1919), in the presence of each of the following carbohydrates, with the following results:

    ______________________________________                                         Basal medium (control) -                                                                             Glycerol +                                               Adonitol -            Inositol +                                               Arabinose +           Lactose -                                                Cellobiose -          Maltose +                                                Erythritol -          Mannitol +                                               Glucose +             Mannose -                                                Melezitose -          Xylose -                                                 Melibose -            Fructose +                                               Raffinose -           Sucrose -                                                Rhamnose -            Sorbitol -                                               Trehalose -                                                                    ______________________________________                                          Legend:                                                                        +: acids formed                                                                -: acids not formed                                                      

Nocardia sp. SC 11,340 decomposes casein and hypoxanthine, but not tyrosine, xanthine and quanine. There is hydrolysis of gelatin, but not starch.

The Antibiotic

The antibiotic EM 4940 can be produced by cultivating Nocardia sp. SC 11,340 A.T.C.C. No. 31531 at, or about, 28° C. under submerged aerobic conditions on an aqueous nutrient medium containing an assimilable carbohydrate and nitrogen source. The fermentation is carried out until substantial antibiotic activity is imparted to the medium, usually about 120 to 144 hours, preferably about 144 hours.

EM 4940 can be separated from the fermentation medium and purified using art-recognized techniques. For example, the broth can be centrifuged to remove the mycelium and then concentrated under reduced pressure. (Alternatively, filtration can be used to remove the mycelium from the broth). Inactive maerial can be precipitated from the concentrate using, for example, methanol, and then discarded. Extraction of the concentrate with ethyl acetate, removes most of the activity. The extract can be concentrated, applied to a chromatography column, e.g., one comprising silicic acid and cellulose, and the antibiotic EM 4940 eluted with chloroform. EM 4940 can be crystallized from acetonitrile. The crystals consist of two forms, (A) platelets and (B) needles. Biologically, the A form is more active than the B form. The two forms are diastereoisomers and cannot usually be distinguished by UV, I.R., mass spectrometry or elemental analysis. One way to distinguish between the two isomers is by proton N.M.R. where the A form has a C--CH₃ peak at 1.75 ppm, while the B form has a C--CH₃ peak at 1.65 ppm. A mixture of A+B forms has both peaks and from integration of the peaks, one can estimate the percentage of each in the sample. Separation of the two forms can be achieved by fractional crystallization from acetonitrile.

EM 4940 (3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyrridinyl)-4-methyl-2H-pyrrole-2-carboxamide) has two assymetric carbon atoms--the carbon atom bearing the carboxamide group and the carbon atom bearing the methyl and hydroxyl groups. Accordingly, 3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide exists in diastereoisomeric forms, or in racemic mixtures thereof. These are all within the scope of this invention.

Detection of EM 4940 is based on a biological diffusion assay utilizing the following medium:

    ______________________________________                                         BBL Seed Agar        30.5   g.                                                 NaCl                 5      g.                                                 Distilled Water      1      liter                                              ______________________________________                                    

The medium is sterilized at 121° C. for 20 minutes. Agar plates containing sub-inhibitory concentrations of diumycin are seeded with E. coli SC 10,888. Under these conditions, EM 4940 is inhibitory to E. coli SC 10,888.

The following examples further illustrate this invention.

EXAMPLE 1

A 250 liter batch of Nocardia sp. SC 11,340 A.T.C.C. No. 31531 is fermented in a 100 gallon stainless steel vessel with the media and operating conditions as described below.

Stage 1: Nocardia sp. SC 11,340 is maintained on the following sterilized agar medium (A):

    ______________________________________                                         Oatmeal                20    g.                                                Tomato paste           20    g.                                                Agar                   15    g.                                                Tap water to 1 liter                                                           ______________________________________                                    

The medium is adjusted to pH 7.0 and sterilized at 121° C. for 15 minutes. A loopful of surface growth from an agar slant (Medium A) of Nocardia sp. SC 11,340 A.T.C.C. No. 31531 is suspended in 11 ml of 0.01% dupanol solution and is used as the source of inoculum to inoculate the following sterilized medium (B).

    ______________________________________                                         Yeast extract           4     g.                                               Malt extract            10    g.                                               Dextrose                4     g.                                               Distilled water to 1 liter                                                     ______________________________________                                    

The medium is adjusted to pH 7.3 and sterilized at 121° C. for 15 minutes. One thousand five hundred ml of this medium in a 4 liter flask is incubated 72 hours on a rotary shaker at 28° C. (300 rpm, 2 inch stroke).

Stage 2 Inoculum: 1.5 liters from stage 1.

Medium B (28.5 liters, as described above) is adjusted to pH 7.3 and sterilized at 121° C. for 15 minutes. To the sterilized medium is added 1.5 liters from stage 1 and the 30 liters of material is incubated for 72 hours. During incubation, the broth is aerated at the rate of 2.3 cubic feed per minute with agitation at 220 rpm.

Stage 3 Inoculum: 12.5 liters from stage 2

Medium B (237.5 liters, as described above) is prepared as in stage 2 and then combined with 12.5 liters of inoculum from stage 2. The resulting 250 liters of material is incubated for 144 hours. During incubation the broth is agitated at 155 rpm and aerated at the rate of 10 cubic feet per minute.

Removal of the mycelium by centrifugation leaves 185 liters of broth. The clear broth is concentrated under reduced pressure at or below 45° C. to about 3.25 liters. The concentrate is poured slowly into 25 liters of methanol with stirring for 30 minutes, the mixture is centrifuged, and the inactive precipitate is discarded. The supernatant is concentrated under reduced pressure at or below 45° C. to approximately 1.8 liters at which point most of the methanol is removed. The concentrate is then extracted four times with 1/2 volume ethyl acetate. The ethyl acetate extracts are combined and concentrated under reduced pressure at or below 45° C. to approximately 100 ml, which contains more than 90% of the activity. The ethyl acetate concentrate is loaded onto a silicic acid/cellulose (2:1 by weight) column (43×70 cm), packed in chloroform.

The column is eluted with chloroform. Approximately 3 liters of active material is collected from the column which after concentrating to dryness gives a dry weight of 11-12 g. This material is crystallized from acetonitrile to yield 4.1 g of crystalline EM 4940.

The crystals of EM 4940 consist of two forms, (A) platelets and (B) needles. Biologically, the A form is more active than the B form. The two forms are diastereoisomers and cannot be distinguished by UV, I.R., mass spectrometry or elemental analysis. One way to distinguish between the two isomers, is by proton N.M.R. where the A form has a C--CH₃ peak at 1.75 ppm, while the B form has a C--CH₃ peak at 1.65 ppm. A mixture of A+B forms has both peaks and from integration of the peaks, one can estimate the percentage of each in the sample. The 4.1 g of EM 4940 obtained above, contains 80% A form and 20% B form. Separation of the two forms is achieved by dissolving the sample in acetonitrile. The B form is more soluble and the A form, therefore, precipitates first; 1.8 g of 97% pure A form is isolated from the sample.

The A form of EM 4940 (platelets) has been determined to be an isomer of 2,4-trans-3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide, and has the following characteristics:

(1) melting point 165°-168° C.

(2) soluble in methanol, ethanol, ethyl acetate, acetone and chloroform Insoluble in hexane and water

(3) mass spectrometry--molecular ion=235; empirical formula=C₁₁ H₁₃ N₃ O₃

(4) elemental analysis

    ______________________________________                                         Element   Found     Calculated for C.sub.11 H.sub.13 N.sub.3 O.sub.3           ______________________________________                                         C         56.14%    56.22                                                      H          5.44%    5.58                                                       N         17.66%    17.88                                                      ash       0                                                                    ______________________________________                                    

(5) optical rotation α_(D) =+20° in methanol

The B form of EM 4940 (needles) has been determined to be an isomer of 2,4-cis-3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide, and has the following characteristics:

(1) melting point 200°-201° C.

(2) mass spectrometry=molecular ion=235; empirical formulal--C₁₁ H₁₃ N₃ O₃

(3) elemental analysis

    ______________________________________                                         Element    Found   Calculated for C.sub.11 H.sub.13 N.sub.3 O.sub.3            ______________________________________                                         C          56.38   56.22                                                       H           5.60   5.58                                                        N          17.78   17.88                                                       ash        0                                                                   ______________________________________                                    

EXAMPLE 2

Nocardia sp. SC 11,340 A.T.C.C. No. 31531 is maintained on the following sterilized agar medium (A):

    ______________________________________                                         Oatmeal                20    g.                                                Tomato paste           20    g.                                                Agar                   15    g.                                                Tap water to 1 liter                                                           ______________________________________                                    

The medium is adjusted to pH 7.0 and autoclaved at 121° C. for 15 minutes.

A loopful of surface growth from an agar slant (medium A) of Nocardia sp. SC 11,340 , (ATCC 31531) is used to inoculate each of six 500 ml Erlenmeyer flasks each containing 100 ml of the following sterilized medium (B).

    ______________________________________                                         Yeast extract           4     g.                                               Malt extract            10    g.                                               Dextrose                4     g.                                               Distilled water to 1 liter                                                     ______________________________________                                    

The medium is adjusted to pH 7.3 before sterilization at 121° C. for 15 minutes. The flasks are then incubated at 28° C. on a rotary shaker (300 rpm; 2 inch stroke) for approximately 72 hours. After the appropriate incubation as described above, 5% (vol/vol) transfers are made from the grown culture flasks to one hundred, 500 ml Erlenmeyer flasks each containing 100 ml of sterilized medium (B) as described above.

After inoculation the flasks are incubated at 28° C. on a rotary shaker (3000 rpm, 2 inch stroke) for approximately 120-144 hours. At this time, the contents of the flasks are pooled and the broth is centrifuged yielding approxmately 9 liters of supernatant broth.

The antibiotic is extracted using the procedure described in Example 1.

In addition to the fermentation media utilized in the above examples, the following media are exemplary of others that can be used for the production of EM 4940 and can be substituted for medium B in the above examples:

    ______________________________________                                         Medium C           Grams                                                       ______________________________________                                         Yeast Extract      4                                                           Malt Extract       10                                                          Dextrose           25                                                          Distilled H.sub.2 O to 1 liter                                                 ______________________________________                                    

pH adjusted to 7.3 before sterilization @121° C. for 15 minutes.

    ______________________________________                                         Medium D           Grams                                                       ______________________________________                                         Glycerol           30                                                          Pharmamedia        20                                                          Yeast Extract      20                                                          Glucose            20                                                          Distilled H.sub.2 O to 1 liter                                                 ______________________________________                                    

pH adjusted to 7.3 before sterilization @121°° C. for 15 minutes.

    ______________________________________                                         Medium E           Grams                                                       ______________________________________                                         Yeast Extract      4                                                           Malt Extract       10                                                          Glucose            10                                                          Glycerol           2                                                           Distilled H.sub.2 O to 1 liter                                                 ______________________________________                                    

pH adjusted to 7.3 before sterilization @121° C. for 15 minutes.

    ______________________________________                                         Medium F          Grams                                                        ______________________________________                                         Glycerol          10                                                           L-Asparagine      5                                                            KH.sub.2 PO.sub.4 1                                                            Na.sub.2 HPO.sub.4                                                                               2                                                            Glucose           50                                                           Mg SO.sub.4 . 7H.sub.2 O                                                                         0.2                                                          Tap H.sub.2 O to 1 liter                                                       ______________________________________                                    

pH adjusted to 7.3 before sterilization @121° C. for 15 minutes.

    ______________________________________                                         Medium G           Grams                                                       ______________________________________                                         (NH.sub.4).sub.2 SO.sub.4                                                                         2                                                           L-Asparagine       5                                                           Glucose            50                                                          Glycerol           10                                                          KH.sub.2 PO.sub.4  7                                                           K.sub.2 HPO.sub.4  7                                                           Mg SO.sub.4 . 7H.sub.2 O                                                                          0.2                                                         Distilled H.sub.2 O to 1 liter                                                 ______________________________________                                    

pH adjusted to 7.3 before sterilization @121° C. for 15 minutes.

BIOLOGICAL TESTING

(A) Minimum Inhibitory Concentration (MIC) determination of EM 4940A and EM 4940B against organisms in an aerobic screen.

1. Using BA-2 Agar

The antibiotic is prepared in 0.1 M phosphate buffer pH 7.0 at a concentration of 100 μg/ml. Two fold dilutions are made in Mueller Hinton broth resulting in a range from 1000 μg/ml to 0.5 μg/ml. One ml of each dilution is placed into individual 100×15 mm petri dishes to which 9.0 ml of BA-2 agar are added. The final drug concentration in the agar ranges from 100 μg/ml to 0.05 μg/ml.

All orgainsms are grown in approximately 15-20 ml of antibiotic assay (AA) broth (Difco Bacto antibiotic medium No. 3) by inoculating the broth with a loopful of the organism from a BHI agar slant. The inoculated tubes are incubated at 37° C. for 18-20 hours. Each incubated broth culture is checked for growth level by a visual turbimetric reading against a McFarland No. 5 standard and adjusted accordingly with AA broth (A McFarland No. 5 standard is equivalent to approximately 1×10⁹ CFU/ml CFU is colony forming units). The visual check is made immediately prior to use. The adjusted broth for each organism is then tested at the appropriated inoculum level. For this series of tests in the inoculum level is 10⁴ CFU. The broth cultures were thus diluted 1:100 in AA broth to give 10⁷ CFU/ml. The organisms are then stamped onto the agar plates using a Denley Multi Point Innoculator which delivers 0.001 ml of culture producing an inoculum level of 10⁴ CFU. After stamping, the plates are incubated at 37° C. for 18 hours. The MIC is recorded as the lowest concentration of antibiotic that inhibits the growth of 3 colonies or less.

The results of this procedure are reported in Table I.

                  TABLE I                                                          ______________________________________                                                    Number in collection                                                                          Minimum Inhibitory                                              of E. R. Squibb                                                                               Concentration(μg/ml)                              Organism   and Sons, Inc. EM4940A                                              ______________________________________                                         Acinetobacter                                                                  calcoaceticus                                                                             8333           50.0                                                 Enterobacter                                                                   cloacae    10,459         >100.0                                               Escherichia                                                                    coli       10,404         100.0                                                Escherichia                                                                    coli       10,857         50.0                                                 Escherichia                                                                    coli       10,896         25.0                                                 Klebsiella                                                                     aerogenes  10,436         100.0                                                Klebsiella                                                                     pneumoniae 11,066         50.0                                                 Proteus                                                                        morganii   9774           50.0                                                 Pseudomonas                                                                    aeruginosa 8754           >100.0                                               Pseudomonas                                                                    aeruginosa 9330           50.0                                                 Pseudomonas                                                                    aeruginosa 9545           25.0                                                 Salmonella                                                                     typhimurium                                                                               9201           50.0                                                 Serratia                                                                       marcescens 9782           100.0                                                Serratia                                                                       marcescens 1111           >100.0                                               Shigella                                                                       sonnei     8449           50.0                                                 Staphylococcus                                                                 aureus     2400           >100.0                                               Staphylococcus                                                                 aureus     10,165         50.0                                                 Staphylococcus                                                                 aureus     11,239         >100.0                                               Streptococcus                                                                  faecalis   9011           >100.0                                               ______________________________________                                    

Additional tests were run using the above methodology and the results of these tests are reported in Table II.

                  TABLE II                                                         ______________________________________                                                 Number in collection                                                                        Minimum Inhibitory                                                of E. R. Squibb                                                                             Concentration(μg/ml)                                   Organism  and Sons, Inc. EM4940A   EM4940B                                     ______________________________________                                         Staphylococcus                                                                 aureus    2400           >200      >200                                        Staphylococcus                                                                 aureus    10,165         100       100                                         Staphylococcus                                                                 aureus    11,239         50        100                                         Staphylococcus                                                                 aureus    2661           >200      >200                                        Streptococcus                                                                  faecalis  9011           >200      >200                                        Escherichia                                                                    coli      10,404         50        50                                          Escherichia                                                                    coli      10,857         50        50                                          Salmonella                                                                     typhimurium                                                                              9201           50        50                                          Klebsiella                                                                     aerogenes 10,436         50        50                                          K. pneumoniae                                                                            11,066         50        50                                          Proteus rettgeri                                                                         8217           50        50                                          P. morganii                                                                              9774           100       100                                         Serratia                                                                       marcescens                                                                               9782           50        50                                          Serratia                                                                       marcescens                                                                               11,111         100       100                                         Pseudomonas                                                                    aeruginosa                                                                               9545           12.5      12.5                                        Pseudomonas                                                                    aeruginosa                                                                               8754           100       100                                         Pseudomonas                                                                    aeruginosa                                                                               9330           >200      >200                                        Acinetobacter                                                                  calcoaceticus                                                                            8333           100       100                                         Enterobacter                                                                   cloacae   10,459         50        100                                         Shigella                                                                       sonnei    8449           50        50                                          ______________________________________                                    

(A) Using H-59 Synthetic Agar

The method is as described above except that H-59 agar is substituted for BA-2 agar.

The results of this procedure are described in Table III.

                  TABLE III                                                        ______________________________________                                                    Number in collection                                                                          Minimum Inhibitory                                              of E. R. Squibb                                                                               Concentration(μ g/ml)                             Organism   and Sons, Inc. EM4940A                                              ______________________________________                                         Acinetobacter                                                                  calcoaceticus                                                                             8333           25.0                                                 Enterobacter                                                                   cloacae    10,459         100.0                                                Escherichia                                                                    coli       10,404         50.0                                                 Escherichia                                                                    coli       10,857         25.0                                                 Klebsiella                                                                     aerogenes  10,436         50.0                                                 Klebsiella                                                                     penumoniae 11,066         50.0                                                 Pseudomonas                                                                    aeruginosa 8754           >100.0                                               Pseudomonas                                                                    aeruginosa 9330           50.0                                                 Pesudomonas                                                                    aeruginosa 9545           25.0                                                 Salmonella                                                                     typhimurium                                                                               9201           50.0                                                 Serratia                                                                       marcescens 9782           100.0                                                Serratia                                                                       marcescens 11,111         100.0                                                Shigella                                                                       sonnei     8449           50.0                                                 Streptococcus                                                                  faecalis   9011           >100.0                                               ______________________________________                                    

(B) Minimum Inhibitory Concentration (MIC) determination of EM4940A against organisms in an anaerobic screen.

The antibiotic is prepared as described above for the aerobic screen.

All organisms are maintained in a tube of chopped meat broth with glucose supplied by Scott Laboratories in Rhode Island. Two days prior to running the test 0.1 ml of the maintained culture is transferred to a fresh tube of chopped meat broth, and the tube is incubated at 37° C. for 48 hours. After 48 hours incubation each culture is assumed to have grown to 1×10⁸ CFU/ml.

The organisms are stamped onto antibiotic containing agar plates (Mueller Hinton Agar BBL+5% whole sheep blood+0.2% lysed sheep blood) to give an inoculum level of 10⁵ CFU.

After stamping, the plates are placed in an anaerobic gas pack system (BBL) and incubated at 37° C. for 18 hours. The MIC is recorded as the lowest concentration of antibiotic that inhibits the growth of 3 colonies or less.

The results of this procedure are reported in Table IV.

                  TABLE IV                                                         ______________________________________                                                    Number in collection                                                                          Minimum Inhibitory                                              of E. R. Squibb                                                                               Concentration(μg/ml)                              Organism   and Sons, Inc. EM4940A                                              ______________________________________                                         Bacteroides                                                                    fragilis     9005         50.00                                                Bacteroides                                                                    fragilis     9844         12.50                                                Bacteroides                                                                    fragilis   10,277         50.00                                                Bacteroides                                                                    fragilis   10,278         50.00                                                Bacteroides                                                                    fragilis   10,279         50.00                                                Bacteroides                                                                    fragilis   10,281         50.00                                                Bacteroides                                                                    fragilis   11,085         100.00                                               Bacteroides                                                                    fragilis   11,086         50.00                                                Bifidobacterium                                                                dentium    11,260         >100.00                                              Clostridium                                                                    histolyticum                                                                                8572         12.50                                                Clostridium                                                                    perfringens                                                                               11,256         50.00                                                Clostridium                                                                    septicum     1780         12.50                                                Clostridium                                                                    sporogenes   2372         12.50                                                Eubacterium                                                                    lentum     11,261         12.50                                                Fusobacterium                                                                  necrophorum                                                                               11,338         12.50                                                Hemophilus                                                                     vaginalis    8568         100.00                                               Hemophilus                                                                     vaginalis    9640         100.00                                               Peptococcus                                                                    variabilis 11,264         50.00                                                Peptostreptococcus                                                             anaerobius 11,263         50.00                                                Propionibacterium                                                              acnes        4020         50.00                                                ______________________________________                                    

(C) Minimum Inhibitory Concentration determination of EM494A against Candida albicans.

The day previous to running the test, an overnight broth culture of Candida albicans in both F4 broth and yeast nitrogen broth (YNB) is prepared. The overnight broth culture contains approximately 1.0×10⁷ CFU/ml. Both broth cultures are diluted 10⁻³ in the respective broths; the antibiotic is prepared at a concentration of 2 mg/ml in 0.1 M phosphate buffer pH 7.0. A 1:8 dilute is of Solution A yields a 1:64 dilution (Solution B).

The test compounds are distributed in 13×100 mm sterile test tubes procedure, using Kahn pipettes (0.2 ml) according to the following schedule:

    ______________________________________                                         Tube    Addition         Concentration                                         ______________________________________                                         1       0.2 ml Stock Solution                                                                             100     μg/ml                                    2       0.1 ml Stock Solution                                                                             50      μg/ml                                    3       0.05 ml Stock Solution                                                                            25      μg/ml                                    4       0.2 ml Solution A (1:8                                                         of stock)          12.5    μg/ml                                    5       0.0 ml Solution A (1:8                                                         of stock)          6.3     μg/ml                                    6       0.05 ml Solution A (1:8                                                        of stock)          3.1     μg/ml                                    7       0.2 ml Solution B (1:64                                                        of stock)          1.6     μg/ml                                    8       0.1 ml Solution B (1:64                                                        of stock)          0.8     μg/ml                                    9       0.05 ml Solution B (1:64                                                       of stock)          0.4     μg/ml                                    10      Positive Growth Control                                                        (no compound)      --                                                  ______________________________________                                    

To each of the above tubes add 4.0 ml of the 10⁻³ dilution of the broth culture to give a final inoculum of 10⁴ CFU/ml.

The tubes are incubated at 37° C. for 18 hours. After this time the results are recorded as: + growth comparable to growth in the control tube; ± growth less than contol tube; - no visible growth.

The MIC is the least concentration of antibiotic giving a negative growth tube. The results of the procedure are reported in Table V.

                  TABLE V                                                          ______________________________________                                                          Minimum Inhibitory                                                             Concentration(μg/ml)                                               Number in collection     Medium: Yeast                                         of E. R. Squibb                                                                               Medium:   Nitrogen Base                                 Organism                                                                               and Sons, Inc. F4        + Glucose                                     ______________________________________                                         Candida                                                                        albicans                                                                               5314           >100      >100                                          ______________________________________                                    

(D) Minimum Inhibitory Concentration determination of EM4940A against dermatophytes.

A 1 mg/ml solution of antibiotic is prepared in 0.1 M phosphate buffer pH 7.0. Two-fold dilutions are made in the broth resulting in a range of antibiotic concentrations from 1000 μg/ml-0.5 μg/ml.

All organisms are grown from shock on F-4 agar slants. Each slant is washed with 10 ml of 0.1 H phosphate buffer pH 7.0. The spore suspension is transferred to a sterile ×4 Morton cap tube and the suspension homogenized.

The prepared agar plates are surface inoculated with 0.2 ml of the appropriate spore suspension. The organism is then streaked across the agar surface using a sterile swab. The inoculated plates are incubated at 26° C. for 48 hours.

The MiC is the highest dilution of each compound series showing no growth. The results of the procedure are reported in Table VI.

                  TABLE VI                                                         ______________________________________                                                    Number in collection                                                                          Minimum Inhibitory                                              of E. R. Squibb                                                                               Concentration(μg/ml)                              Organism   and Sons, Inc. EM4940A                                              ______________________________________                                         T. mentagrophytes                                                                         2637           >100                                                 E. floccosum                                                                              9185           >100                                                 T. rubrum  9199             50                                                 M. canis   9237            100                                                 ______________________________________                                    

(E) Minimum Inhibitory Concentration determination of EM4940A against Trichomonas vaginalis.

Compound preparation is carried out as described under (C). To each series of dilutions, 4 ml of a 1:100 dilution of T. vaginalis (grown in modified Diamond's Medium pH 6.0+10% rabbit serum) is made from a 40 hour culture.

The tubes are incubated anaerobically at 37° C. for 40 hours and the MIC's determined. The results of the procedure are reported in Table VII.

                  TABLE VII                                                        ______________________________________                                                   Number in collection                                                                           Minimum Inhibitory                                             of E. R. Squibb Concentration(μg/ml)                              Organism  and Sons, Inc.  EM4940A                                              ______________________________________                                         Trichomonas                                                                    vaginalis 8560            12.5                                                 ______________________________________                                    

(F) Minimum Inhibitory Concentration determination of EM4940A against mycoplasmas.

The antibiotic (1 mg) is dissolved in 200 microliters of dimethylsulfoxide (DMSO). Doubling dilutions are carried out in DMSO over twelve levels to give concentrations ranging from 500-2.49 μg/ml. To two sets of microtitration plates, 5 microliter aliquots of each concentration are transferred in triplicate. Each well of a triplicate set of dilutions is inoculated with 250 microliters of inocultated Channocks broth (10% of 48 hour culture of either M. mycoides or A. laidlawii). Plates are sealed and examined after 48 hours at 37° C.--for phenol red color change red to yellow and the MIC recorded as the lowest concentration in which no color change occurs.

The results of the procedure are reported in Table VIII.

                  TABLE VIII                                                       ______________________________________                                         Organism       MIC (μg/ml) of EM4940A                                       ______________________________________                                         Mycoplasma mycoides                                                                           >100                                                            Acholeplasma laidlawii                                                                        100                                                             ______________________________________                                    

As shown by the above experiments the antibiotics of this invention have activity against a range of gram-positive and gram-negative bacteria, yeasts, fungi, acholeplasma, and the protozoan Tichomonas vaginalis. The antibiotics can, therefore, be used as (1) an environmental disinfectant, (e.g., as a spray or dust in a conventional carrier) and (2) an agent to combat infections due to the above-enumerated bacteria, yeasts, fungi, acholesplasma and Trichomonas vaginalis in various mammalian species (e.g., topically, orally or parenterally using pharmaceutically acceptable carriers, excipients, etc.). 

What is claimed is:
 1. A process for the preparation of the compound 3,4-dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide which comprises cultivating Nocardia sp. 11,340 A.T.C.C. No. 31531 in an aqueous nutrient medium comprising an assimilable carbohydrate and an assimilable nitrogen source under submerged aerobic conditions until substantial antibiotic activity is imparted to the medium.
 2. A process in accordance with claim 1 wherein the microorganism is cultivated at about 28° C. 